The principal goal of this proposal is to use molecular and immunological techniques to develop more sensitive and specific tests for the diagnosis of Lyme borreliosis. To this end, this project is a logical extension of the work we have been performing. In this project we will use specimens of well characterized patients with Lyme disease. We have developed a network in which specimens are prospectively and longitudinally collected from patients in whom detailed case report forms are available. The diagnostic tests which we will develop can be categorized into two groups: 1) To use recombinant proteins and peptides to characterize the humoral response to this pathogen and to utilize these recombinants for the development of a recombinant based assay; 2) To utilize molecular and immunological techniques to identify the pathogen or its product in clinical specimens. The OspA, p41, p66, p73 and p93 have all been cloned by using recombinant techniques. We have mapped the epitopes of OspA and partially mapped the epitopes of p41. We have expressed the p66 protein in EscherichiA coli and plan to map the epitopes. The genes encoding p93 and p73 are partially sequenced, they will be completely sequenced, and the epitopes mapped. In addition, we will continue to define the humoral response to the OspA antigen which is associated with the development of chronic disease. A T7 based expression system will be used to produce in E. coli immunodominant peptides specific to B. burgdorferi. These products will be purified to homogeneity using an effective, general approach for isolation of recombinant proteins and used to develop highly specific immunoassays. We have developed a genomic library based on the pUCphoA expression vector to identify additional surface or secreted (exoantigens?) and continue to use a lambda gtll library to identify proteins which may be potentially useful for diagnosis of Lyme disease.